Mesenchymal stem cell: Basic research and potential applications in cattle and buffalo

Characteristic features like self-renewal, multilineage differentiation potential, and immune-modulatory/anti-inflammatory properties, besides the ability to mobilize and home distant tissues make stem cells (SCs) a lifeline for an individual. Stem cells (SCs) if could be harvested and expanded without any abnormal change may be utilized as an all-in-one solution to numerous clinical ailments. However, slender understanding of their basic physiological properties, including expression potential, behavioral alternations during culture, and the effect of niche/microenvironment has currently restricted the clinical application of SCs. Among various types of SCs, mesenchymal stem cells (MSCs) are extensively studied due to their easy availability, straightforward harvesting, and culturing procedures, besides, their less likelihood to produce teratogens. Large ruminant MSCs have been harvested from various adult tissues and fetal membranes and are well characterized under in vitro conditions but unlike human or other domestic animals in vivo studies on cattle/buffalo MSCs have mostly been aimed at improving the animals’ production potential. In this document, we focused on the status and potential application of MSCs in cattle and buffalo.     

Mesenchymal stem cells: a double-edged sword in regulating immune responses

Mesenchymal stem cells (MSCs) have been employed successfully to treat various immune disorders in animal models and clinical settings. Our previous studies have shown that MSCs can become highly immunosuppressive upon stimulation by inflammatory cytokines, an effect exerted through the concerted action of chemokines and nitric oxide (NO). Here, we show that MSCs can also enhance immune responses. This immune-promoting effect occurred when proinflammatory cytokines were inadequate to elicit sufficient NO production. When inducible nitric oxide synthase (iNOS) production was inhibited or genetically ablated, MSCs strongly enhance T-cell proliferation in vitro and the delayed-type hypersensitivity response in vivo. Furthermore, iNOS(-/-) MSCs significantly inhibited melanoma growth. It is likely that in the absence of NO, chemokines act to promote immune responses. Indeed, in CCR5(-/-)CXCR3(-/-) mice, the immune-promoting effect of iNOS(-/-) MSCs is greatly diminished. Thus, NO acts as a switch in MSC-mediated immunomodulation. More importantly, the dual effect on immune reactions was also observed in human MSCs, in which indoleamine 2,3-dioxygenase (IDO) acts as a switch. This study provides novel information about the pathophysiological roles of MSCs.     

Pluripotent stem cell-derived neural stem cells: From basic research to applications

Basic research on pluripotent stem cells is designed to enhance understanding of embryogenesis, whereas applied research is designed to develop novel therapies and prevent diseases. Attainment of these goals has been enhanced by the establishment of embryonic stem cell lines, the technological development of genomic reprogramming to generate induced-pluripotent stem cells, and improvements in vitro techniques to manipulate stem cells. This review summarizes the techniques required to generate neural cells from pluripotent stem cells. In particular, this review describes current research applications of a simple neural differentiation method, the neural stem sphere method, which we developed.     

WJSC 6th Anniversary Special Issues（2）:Mesenchymal stem cells Mesenchymal stem cells in the treatment of spinal cord injuries:A review

With technological advances in basic research,the intricate mechanism of secondary delayed spinal cord injury(SCI)continues to unravel at a rapid pace.However,despite our deeper understanding of the molecular changes occurring after initial insult to the spinal cord,the cure for paralysis remains elusive.Current treatment of SCI is limited to early administration of high dose steroids to mitigate the harmful effect of cord edema that occurs after SCI and to reduce the cascade of secondary delayed SCI.R ecent evident-based clinical studies have cast doubt on the clinical benefit of steroids in SCI and intense focus on stem cell-based therapy has yielded some encouraging results.An array of mesenchymal stem cells(MSCs)from various sources with novel and promising strategies are being developed to improve function after SCI.In this review,we briefly discuss the pathophysiology of spinal cord injuries and characteristics and the potential sources of MSCs that can be used in the treatment of SCI.We will discuss the progress of MSCs application in research,focusing on the neuroprotective properties of MSCs.Finally,we will discuss the results from preclinical and clinical trials involving stem cell-based therapy in SCI.     

Progress in the use of dental pulp stem cells in regenerative medicine

The field of tissue engineering is emerging as a multidisciplinary area with promising potential for regenerating new tissues and organs. This approach requires the involvement of three essential components: stem cells, scaffolds and growth factors. To date, dental pulp stem cells have received special attention because they represent a readily accessible source of stem cells. Their high plasticity and multipotential capacity to differentiate into a large array of tissues can be explained by its neural crest origin, which supports applications beyond the scope of oral tissues. Many isolation, culture and cryopreservation protocols have been proposed that are known to affect cell phenotype, proliferation rate and differentiation capacity. The clinical applications of therapies based on dental pulp stem cells demand the development of new biomaterials suitable for regenerative purposes that can act as scaffolds to handle, carry and implant stem cells into patients. Currently, the development of xeno-free culture media is emerging as a means of standardization to improve safe and reproducibility. The present review aims to describe the current knowledge of dental pulp stem cells, considering in depth the key aspects related to the characterization, establishment, maintenance and cryopreservation of primary cultures and their involvement in the multilineage differentiation potential. The main clinical applications for these stem cells and their combination with several biomaterials is also covered.     

Mesenchymal stem cell therapy of brain ischemic stroke in rats

Mesenchymal stem cell (MSCs)-based therapy is a promising attempt to improve the recovery after stroke. Our experiments were carried out on inbred Wistar-Kyoto rats. MSCs were isolated, expanded in culture, and labeled with vital fluorescent dye PKH-26. Animals were subjected to middle cerebral artery occlusion (MCAO). After three days, MCAO 5 × 10⁶ isolated MSCs were injected into the tail vein of the experimental rats. The control animal group received PBS injections (negative control). Therapy results were evaluated by the following parameters: behavioral and neurological testing, the inured brain areas, damaged brain structures, neuron state, and vessel quantity in the region close to with necrosis zone. It was shown that control animals (PBS injection) did not return to their initial behavioral and neurological state within 6 weeks, while the experimental animals (MSCs injection), within 2–3 weeks after MCAO, had parameters like intact rats. The size of the damaged region in the control group was larger than in the experimental group by a factor of approximately 1.3. The damage in MSC-treated rats was limited to the neocortex; caudate nucleus, capsula externa and piriform cortex remained uninjured. The small vessel quantity in the “border” regions was twice as high as compared to the control group and approximately equal to the number of vessels in an intact brain. For the first time, we demonstrated that the vessel quantity in the neocortex and caudate nucleus of the contralateral hemisphere after MSC transplantation was twice as high as in control rats. It is concluded that the MSC transplantation exerts a beneficial influence upon the brain tissue reparation after stroke.     

Adipose‐derived mesenchymal stem cells and regenerative medicine

Adipose tissue‐derived mesenchymal stem cells (ADSCs) are multipotent and can differentiate into various cell types, including osteocytes, adipocytes, neural cells, vascular endothelial cells, cardiomyocytes, pancreatic β‐cells, and hepatocytes. Compared with the extraction of other stem cells such as bone marrow‐derived mesenchymal stem cells (BMSCs), that of ADSCs requires minimally invasive techniques. In the field of regenerative medicine, the use of autologous cells is preferable to embryonic stem cells or induced pluripotent stem cells. Therefore, ADSCs are a useful resource for drug screening and regenerative medicine. Here we present the methods and mechanisms underlying the induction of multilineage cells from ADSCs.     

Stem cell therapies and regenerative medicine in China

Stem cells are the core of tissue repair and regeneration,and a promising cell source for novel therapies.In recent years,research into stem cell therapies has been particularly exciting in China.The remarkable advancements in basic stem cell research and clinically effective trials have led to fresh insights into regenerative medicine,such as treatments for sweat gland injury after burns,diabetes,and liver injury.High hopes have inspired numerous experimental and clinical trials.At the same time,government investment and policy support of research continues to increase markedly.However,numerous challenges must be overcome before novel stem cell therapies can achieve meaningful clinical outcomes.     

Mesenchymal stem cell modification of endothelial matrix regulates their vascular differentiation

Mesenchymal stem cells (MSCs) respond to a variety of differentiation signal provided by their local environments. A large portion of these signals originate from the extracellular matrix (ECM). At the same time, MSCs secrete various matrix‐altering agents, including proteases, that alter ECM‐encoded differentiation signals. Here we investigated the interactions between MSC and ECM produced by endothelial cells (EC‐matrix), focusing not only on the differentiation signals provided by EC‐matrix, but also on MSC‐alteration of these signals and the resultant affects on MSC differentiation. MSCs were cultured on EC‐matrix modified in one of three distinct ways. First, MSCs cultured on native EC‐matrix underwent endothelial cell (EC) differentiation early during the culture period and smooth muscle cell (SMC) differentiation at later time points. Second, MSCs cultured on crosslinked EC‐matrix, which is resistant to MSC modification, differentiated towards an EC lineage only. Third, MSCs cultured on EC‐matrix pre‐modified by MSCs underwent SMC‐differentiation only. These MSC‐induced matrix alterations were found to deplete the factors responsible for EC‐differentiation, yet activate the SMC‐differentiation factors. In conclusion, our results demonstrate that the EC‐matrix contains factors that support MSC differentiation into both ECs and SMCs, and that these factors are modified by MSC‐secreted agents. By analyzing the framework by which EC‐matrix regulates differentiation in MSCs, we have uncovered evidence of a feedback system in which MSCs are able to alter the very matrix signals acting upon them. J. Cell. Biochem. 107: 706–713, 2009. Published 2009 Wiley‐Liss, Inc.     

Mesenchymal stem cell mediated effects on microglial phenotype in cuprizone-induced demyelination model

Microglial cells have an essential role in neurodegenerative disorders, such as multiple sclerosis. They are divided into two subgroups: M1 and M2 phenotypes. Mesenchymal stem cells (MSC), with neuroprotective and immunomodulating properties, could improve these diseases. We evaluate the immunomodulating effects of MSC on microglial phenotypes and the improvement of demyelination in a cuprizone (CPZ) model of multiple sclerosis (MS). For inducing the chronic demyelination model, C57BL6 mice were given a diet with 0.2% CPZ (w/w) for 12 weeks. In the MSC group, cells were transplanted into the right lateral ventricle of mice. The expression of targeted genes was assessed by real-time polymerase chain reaction. M1 and M2 microglial phenotypes were assessed by immunohistochemistry of inducible nitric oxide synthase (iNOS) and Arg-1, respectively. Remyelination was studied by luxal fast blue (LFB) staining and electron microscopy (EM). We found that MSC transplantation reduced the expression level of M1-specific messenger RNA (mRNA; iNOS and CD86) but increased the expression level of M2 specific genes (CD206, Arg-1, and CX3CR1) in comparison to the CPZ group. Moreover, cell therapy significantly decreased the M1 marker (iNOS⁺ cells), but M2 marker (Arg-1⁺ cells) significantly increased in comparison with the CPZ group. In addition, MSC treatment significantly increased the CX3CL1 expression level in comparison with the CPZ group and led to improvement in remyelination, which was confirmed by LFB and EM images. The results showed that MSC transplantation increases the M2 and decreases the M1 phenotype in MS. This change was accompanied by decrease in demyelination and axonal injury and indicated that MSCs have a positive effect on MS by modification of microglia cells.     

Priming mesenchymal stem cells boosts stem cell therapy to treat myocardial infarction

Cardiovascular diseases are the number one cause of death globally and are projected to remain the single leading cause of death. Treatment options abounds, although efficacy is limited. Recent studies attribute discrete and ephemeral benefits to adult stem cell therapies, indicating the urge to improve stem cell based–therapy. In this study, we show that priming mesenchymal stem cells (MSC) towards cardiomyogenic lineage enhances their beneficial effects in vivo as treatment option for acute phase myocardial infarction. MSC were primed using cardiomyogenic media for 4 days, after which peak expression of key cardiomyogenic genes are reached and protein expression of Cx‐43 and sarcomeric α‐actinin are observed. MSC and primed MSC (pMSC) were characterized in vitro and used to treat infarcted rats immediately after left anterior descending (LAD) occlusion. Echocardiography analysis indicated that MSC‐treated myocardium presented discrete improvement in function, but it also showed that pMSC treatment lead to superior beneficial results, compared with undifferentiated MSC. Seven days after cell injection, MSC and pMSC could still be detected in the myocardium. Connexin‐43 expression was quantified through immunoblotting, and was superior in pMSC, indicating that this could be a possible explanation for the superior performance of pMSC therapy.     

Mesenchymal stem cells differentiate into keratinocytes and express epidermal kallikreins: Towards an in vitro model of human epidermis

Epidermal differentiation is a complex process in which keratinocytes go through morphological and biochemical changes in approximately 15 to 30 days. Abnormal keratinocyte differentiation is involved in the pathophysiology of several skin diseases. In this scenario, mesenchymal stem cells (MSCs) emerge as a promising approach to study skin biology in both normal and pathological conditions. Herein, we have studied the differentiation of MSC from umbilical cord into keratinocytes. MSC were cultured in Dulbecco's modified Eagle's medium (DMEM) (proliferation medium) and, after characterization, differentiation was induced by culturing cells in a defined keratinocyte serum-free medium (KSFM) supplemented with epidermal growth factor (EGF) and calcium chloride ions. Cells cultivated in DMEM were used as control. Cultures were evaluated from day 1 to 23, based on the cell morphology, the expression of p63, involucrin and cytokeratins (KRTs) KRT5, KRT10 and KRT14, by quantitative polymerase chain reaction, Western blot analysis or immunofluorescence, and by the detection of epidermal kallikreins activity. In cells grown in keratinocyte serum-free medium with EGF and 1.8 mM calcium, KRT5 and KRT14 expression was shown at the first day, followed by the expression of p63 at the seventh day. KRT10 expression was detected from day seventh while involucrin was observed after this period. Data showed higher kallikrein (KLK) activity in KSFM-cultured cells from day 11th in comparison to control. These data indicate that MSC differentiated into keratinocytes similarly to that occurs in the human epidermis. KLK activity detection appears to be a good methodology for the monitoring the differentiation of MSC into the keratinocyte lineage, providing useful tools for the better understanding of the skin biology.     

Wharton’s jelly‐derived stromal cells and their cell therapy applications in allogeneic haematopoietic stem cell transplantation

For decades, mesenchymal stromal cells (MSCs) have been of great interest in the fields of regenerative medicine, tissue engineering and immunomodulation. Their tremendous potential makes it desirable to cryopreserve and bank MSCs to increase their accessibility and availability. Postnatally derived MSCs seem to be of particular interest because they are harvested after delivery without ethical controversy, they have the capacity to expand at a higher rate than adult‐derived MSCs, in which expansion decreases with ageing, and they have demonstrated immunological and haematological supportive properties similar to those of adult‐derived MSCs. In this review, we focus on MSCs obtained from Wharton''s jelly (the mucous connective tissue of the umbilical cord between the amniotic epithelium and the umbilical vessels). Wharton''s jelly MSCs (WJ‐MSCs) are a good candidate for cellular therapy in haematology, with accumulating data supporting their potential to sustain haematopoietic stem cell engraftment and to modulate alloreactivity such as Graft Versus Host Disease (GVHD). We first present an overview of their in‐vitro properties and the results of preclinical murine models confirming the suitability of WJ‐MSCs for cellular therapy in haematology. Next, we focus on clinical trials and discuss tolerance, efficacy and infusion protocols reported in haematology for GVHD and engraftment.     

Mesenchymal stem cells as a therapeutic tool to treat sepsis

Sepsis is a clinical syndrome caused by a deregulated host response to an infection. Sepsis is the most frequent cause of death in hospitalized patients. Although knowledge of the pathogenesis of sepsis has increased substantially during the last decades, attempts to design effective and specific therapiestargeting components of the derailed host response have failed. Therefore, there is a dramatic need for new and mechanistically alternative therapies to treat this syndrome. Based on their immunomodulatory properties, adult mesenchymal stem or stromal cells(MSCs) can be a novel therapeutic tool to treat sepsis. Indeed, MSCs reduce mortality in experimental models of sepsis by modulating the deregulated inflammatory response against bacteria through the regulation of multiple inflammatory networks, the reprogramming of macrophages and neutrophils towards a more antiinflammatory phenotype and the release of antimicrobial peptides. This report will review the current knowledge on the effects of MSC treatment in preclinical experimental small animal models of sepsis.     

Mesenchymal stem cells show radioresistance in vivo

Irradiation impacts on the viability and differentiation capacity of tissue‐borne mesenchymal stem cells (MSC), which play a pivotal role in bone regeneration. As a consequence of radiotherapy, bones may develop osteoradionecrosis. When irradiating human bone‐derived MSC in vitro with increasing doses, the cells’ self‐renewal capabilities were greatly reduced. Mitotically stalled cells were still capable of differentiating into osteoblasts and pre‐adipocytes. As a large animal model comparable to the clinical situation, pig mandibles were subjected to fractionized radiation of 2 χ 9 Gy within 1 week. This treatment mimics that of a standardized clinical treatment regimen of head and neck cancer patients irradiated 30 χ 2 Gy. In the pig model, fractures which had been irradiated, showed delayed osseous healing. When isolating MSC at different time points post‐irradiation, no significant changes regarding proliferation capacity and osteogenic differentiation potential became apparent. Therefore, pig mandibles were irradiated with a single dose of either 9 or 18 Gy in vivo, and MSC were isolated immediately afterwards. No significant differences between the untreated and 9 Gy irradiated bone with respect to proliferation and osteogenic differentiation were unveiled. Yet, cells isolated from 18 Gy irradiated specimens exhibited a reduced osteogenic differentiation capacity, and during the first 2 weeks proliferation rates were greatly diminished. Thereafter, cells recovered and showed normal proliferation behaviour. These findings imply that MSC can effectively cope with irradiation up to high doses in vivo. This finding should thus be implemented in future therapeutic concepts to protect regenerating tissue from radiation consequences.     

Clinical grade adult stem cell banking

There has been a great deal of scientific interest recently generated by the potential therapeutic applications of adult stem cells in human care but there are several challenges regarding quality and safety in clinical applications and a number of these challenges relate to the processing and banking of these cells ex-vivo. As the number of clinical trials and the variety of adult cells used in regenerative therapy increases, safety remains a primary concern. This has inspired many nations to formulate guidelines and standards for the quality of stem cell collection, processing, testing, banking, packaging and distribution. Clinically applicable cryopreservation and banking of adult stem cells offers unique opportunities to advance the potential uses and widespread implementation of these cells in clinical applications. Most current cryopreservation protocols include animal serum proteins and potentially toxic cryoprotectant additives (CPAs) that prevent direct use of these cells in human therapeutic applications. Long term cryopreservation of adult stem cells under good manufacturing conditions using animal product free solutions is critical to the widespread clinical implementation of ex-vivo adult stem cell therapies. Furthermore, to avoid any potential cryoprotectant related complications, reduced CPA concentrations and efficient post-thaw washing to remove CPA are also desirable. The present review focuses on the current strategies and important aspects of adult stem cell banking for clinical applications. These include current good manufacturing practices (cGMPs), animal protein free freezing solutions, cryoprotectants, freezing & thawing protocols, viability assays, packaging and distribution. The importance and benefits of banking clinical grade adult stem cells are also discussed.